CN109490056B - Cell three-dimensional culture matrigel wax embedding method - Google Patents
Cell three-dimensional culture matrigel wax embedding method Download PDFInfo
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- CN109490056B CN109490056B CN201811297773.5A CN201811297773A CN109490056B CN 109490056 B CN109490056 B CN 109490056B CN 201811297773 A CN201811297773 A CN 201811297773A CN 109490056 B CN109490056 B CN 109490056B
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- tissue
- dimensional culture
- matrigel
- embedding
- slide
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- 108010082117 matrigel Proteins 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000011521 glass Substances 0.000 claims abstract description 12
- 229930040373 Paraformaldehyde Natural products 0.000 claims abstract description 8
- 229920002866 paraformaldehyde Polymers 0.000 claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 claims abstract description 5
- 108010035532 Collagen Proteins 0.000 claims abstract description 4
- 102000008186 Collagen Human genes 0.000 claims abstract description 4
- 229920001436 collagen Polymers 0.000 claims abstract description 4
- 238000007598 dipping method Methods 0.000 claims abstract description 4
- 229920006324 polyoxymethylene Polymers 0.000 claims abstract description 4
- 239000003292 glue Substances 0.000 claims abstract 5
- 239000011159 matrix material Substances 0.000 claims abstract 5
- 230000018044 dehydration Effects 0.000 claims description 5
- 238000006297 dehydration reaction Methods 0.000 claims description 5
- 239000001993 wax Substances 0.000 description 13
- 239000000853 adhesive Substances 0.000 description 5
- 230000001070 adhesive effect Effects 0.000 description 5
- 230000007547 defect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a cell three-dimensional culture matrix glue wax embedding method, which comprises the following steps: (1) the embedding box is used for designing and manufacturing a glass slide matched with the shape of the internal accommodating space of the embedding box; (2) cutting the matrigel three-dimensional culture tissue from the upper chamber of a Transwell, and fixing in paraformaldehyde with the mass percentage of 4-6% for 6-24 hours; (3) after the fixation is successful, fishing out the matrigel three-dimensional cultured tissue from polyformaldehyde, and adsorbing the collagen surface of the matrigel three-dimensional cultured tissue on a glass sheet; (4) placing the glass slide which is matched with the shape of the internal accommodating space of the embedding box and adsorbs the matrigel three-dimensional culture tissue into the embedding box paved with gauze; (5) dehydrating, transparent and wax-dipping; (6) the tissue adhered to the slide was scraped off intact from the slide with a tissue slicer, and the tissue was embedded. The tissue does not curl, fall off or drop off during the embedding process, and the method is suitable for popularization and application.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a cell three-dimensional culture matrigel wax embedding method.
Background
The biological tissue embedding method is a common method in the field of biotechnology. The traditional embedding method comprises paraffin embedding and plastic embedding. The former has a long production process (about 2 days). The lipid component in the tissue is not easy to be reserved, the tissue cell is easy to shrink, the activity of most enzymes is adversely affected, and the source floating staining cannot be carried out; the latter has the disadvantages that immunohistochemical staining cannot be carried out and expensive matching equipment is required. Although the cryostat used in the seventies has a high speed and can perform both enzymatic and immunohistochemical staining, it is difficult to make thinner slices of 5 μm or less and continuous slices, and it has a defect that the cell structure is unclear due to freezing.
The prior cell three-dimensional culture matrigel wax embedding method has the following defects:
1. matrigel tends to curl during dehydration due to loose texture.
2. The tissue is easy to fall off and lose in the processes of dehydration, transparence and wax soaking.
3. The superficial cells of the tissue are easy to fall off, thereby reducing the observation speed and the efficiency of researching the tissue lesion.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, adapt to practical development and provide an improved cell three-dimensional culture matrigel wax embedding method.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a cell three-dimensional culture matrigel wax embedding method comprises the following steps:
(1) taking a conventional embedding box in the prior art, designing and manufacturing a slide matched with the shape of an internal accommodating space of the embedding box;
(2) cutting the matrigel three-dimensional culture tissue from the upper chamber of a Transwell, and fixing in paraformaldehyde with the mass percentage of 4-6% for 6-24 hours;
(3) after the fixation is successful, fishing out the matrigel three-dimensional culture tissue from polyformaldehyde, and adsorbing the collagen surface of the matrigel three-dimensional culture tissue on the glass sheet;
(4) placing the glass slide which is matched with the shape of the internal accommodating space of the embedding box and adsorbs the matrigel three-dimensional culture tissue into the embedding box paved with gauze;
(5) conventional dehydration, transparency and wax dipping;
(6) the tissue adhered to the slide was scraped off intact from the slide with a tissue microtome, and the tissue was embedded routinely.
Preferably, the slide is an adhesive slide or a normal cover slip.
Preferably, the thickness of the slide is 0.1 mm.
Preferably, the mass percentage of the paraformaldehyde is 5%.
Preferably, the fixing time of the matrigel three-dimensional cultured tissue in paraformaldehyde is 12 hours.
Preferably, the thickness of the gauze paved in the embedding box is 1-3 mm.
Compared with the prior art, the invention has the following advantages and positive effects:
1. solves the problems of tissue curling, shedding and cell dropping in the wax embedding process of the matrigel three-dimensional cultured tissue, and leads the wax embedding of the matrigel three-dimensional cultured tissue to be well realized.
2. Greatly improves the success rate of the matrigel three-dimensional culture tissue slice and increases the diversity of the experimental detection method.
Drawings
FIG. 1 is a schematic view showing the overall structure of an embedding cassette used in an embodiment of the present invention;
FIG. 2 is a schematic structural view of an adherent slide used in the embodiment of the present invention;
the notations in FIGS. 1-2 are as follows:
1. embedding box, 2, fixed slot, 3, fixed knot, 4, connection piece, 5, embedding box apron, 6, buckle, 7, drainage mouth, 8, draw-in groove, 9, hasp, 10, adhesion slide.
Detailed Description
The following is a detailed description of specific embodiments of the invention.
A cell three-dimensional culture matrigel wax embedding method comprises the following steps:
(1) taking a conventional embedding box in the prior art, processing the embedding box by opening a side hole, wherein the structure is shown in figure 1, so that various reagents can fully enter the embedding box after an adhesive slide is covered in the embedding box; designing and manufacturing an adhesive slide matched with the shape of the inner accommodating space of the embedding box, wherein the structure of the adhesive slide is shown in figure 2; the thickness of the slide is 0.1mm, which is thinner than the thickness of the conventional slide in the market by 0.2mm, and the function of the slide is to prevent the slide from being too heavy to deform the three-dimensional culture tissue;
(2) cutting the matrigel three-dimensional culture tissue from the upper chamber of a Transwell, and fixing in paraformaldehyde with the mass percent of 5% for 12 hours;
(3) after the fixation is successful, fishing out the matrigel three-dimensional culture tissue from polyformaldehyde, and adsorbing the collagen surface of the matrigel three-dimensional culture tissue on the adhesive glass sheet;
(4) placing the glass slide which is matched with the shape of the internal accommodating space of the embedding box and adsorbs the matrigel three-dimensional culture tissue into the embedding box paved with gauze with the thickness of 2 mm;
(5) conventional dehydration, transparency and wax dipping;
(6) the tissue adhered to the slide was scraped off intact from the slide with a tissue microtome, and the tissue was embedded routinely.
Through observation and record of laboratory research personnel, the tissues are not curled, fall off or drop off, cells are not dropped off in the wax embedding process of the matrigel three-dimensional culture tissues, the wax embedding of the matrigel three-dimensional culture tissues is ideal, the success rate of the matrigel three-dimensional culture tissue slices is greatly improved, and the diversity of experimental detection methods is increased.
The method has simple operation steps and is suitable for popularization and use in conventional laboratories. Meanwhile, the embedded biological tissue sample can be applied to the technology of combining block-surface imaging with continuous thin slicing, and then relevant structure and function research is carried out.
The above embodiments are merely preferred embodiments of the present invention, and any simple modification, modification and substitution changes made to the above embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
Claims (4)
1. A cell three-dimensional culture matrix glue wax embedding method is characterized in that: the method comprises the following steps:
(1) taking a conventional embedding box in the prior art, designing and manufacturing a glass slide matched with the shape of an internal accommodating space of the embedding box, wherein the thickness of the glass slide is 0.1mm, so as to prevent the glass slide from being overweight to deform the three-dimensional culture tissue;
(2) cutting the matrigel three-dimensional culture tissue from the upper chamber of a Transwell, and fixing in paraformaldehyde with the mass percentage of 4-6% for 6-24 hours;
(3) after the fixation is successful, fishing out the matrigel three-dimensional culture tissue from polyformaldehyde, and adsorbing the collagen surface of the matrigel three-dimensional culture tissue on the glass sheet;
(4) placing the glass slide which is matched with the shape of the internal accommodating space of the embedding box and adsorbs the matrigel three-dimensional culture tissue into the embedding box paved with gauze;
(5) conventional dehydration, transparency and wax dipping;
(6) the tissue adhered to the slide was scraped off intact from the slide with a tissue microtome, and the tissue was embedded routinely.
2. The method for embedding the cell three-dimensional culture matrix glue in the wax according to claim 1, wherein the method comprises the following steps: the mass percent of the paraformaldehyde is 5%.
3. The method for embedding the cell three-dimensional culture matrix glue in the wax according to claim 1, wherein the method comprises the following steps: the fixing time of the matrigel three-dimensional culture tissue in paraformaldehyde is 12 hours.
4. The method for embedding the cell three-dimensional culture matrix glue in the wax according to claim 1, wherein the method comprises the following steps: the thickness of the gauze paved in the embedding box is 1-3 mm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811297773.5A CN109490056B (en) | 2018-10-31 | 2018-10-31 | Cell three-dimensional culture matrigel wax embedding method |
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| CN201811297773.5A CN109490056B (en) | 2018-10-31 | 2018-10-31 | Cell three-dimensional culture matrigel wax embedding method |
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| CN109490056B true CN109490056B (en) | 2021-07-30 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201793576U (en) * | 2010-08-19 | 2011-04-13 | 西北工业大学 | Novel cell culture device |
| CN103509711A (en) * | 2012-06-27 | 2014-01-15 | 毛静涛 | Compound type biological tissue and cell chip |
| CN104853843A (en) * | 2012-09-12 | 2015-08-19 | 比欧帕斯自动化公司 | Microtome sectionable gel support structure and methods |
| CN104977202A (en) * | 2015-08-07 | 2015-10-14 | 广西医科大学 | Preparation method of transmission electron microscope sample of paraffin-embedded section tissue |
| CN107217039A (en) * | 2017-08-01 | 2017-09-29 | 世翱（上海）生物医药科技有限公司 | Tumor tissues 3D cultural methods and nutrient solution |
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- 2018-10-31 CN CN201811297773.5A patent/CN109490056B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201793576U (en) * | 2010-08-19 | 2011-04-13 | 西北工业大学 | Novel cell culture device |
| CN103509711A (en) * | 2012-06-27 | 2014-01-15 | 毛静涛 | Compound type biological tissue and cell chip |
| CN104853843A (en) * | 2012-09-12 | 2015-08-19 | 比欧帕斯自动化公司 | Microtome sectionable gel support structure and methods |
| CN104977202A (en) * | 2015-08-07 | 2015-10-14 | 广西医科大学 | Preparation method of transmission electron microscope sample of paraffin-embedded section tissue |
| CN107217039A (en) * | 2017-08-01 | 2017-09-29 | 世翱（上海）生物医药科技有限公司 | Tumor tissues 3D cultural methods and nutrient solution |
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